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Abstract

Abstract

Nucleoside transporters play an important role in regulating the extracellular concentration of Adenosine and salvaging nucleosides. They also play an essential role in the transport of anticancer and antiviral drugs. Nucleoside transporters have been classified into families known as Equiliberative (ENT) and Concentrative (CNT).

ENTs belong to the SLC 29 transmembrane protein family, with 11 transmembrane domains with intracellular N- terminus and extracellular C terminus. In order to localize to the plasma membrane they must undergo correct processing, targeting and trafficking. Constructs were designed where the intracellular and extracellular loops were deleted and used to test the efficiency of Adenosine and Uridine transport in Xenopus laevis oocytes using radiolabelled substrates.

Stage VI Xenopus oocytes were injected with 23ng hENT1 RNA, and incubated for 48 hrs at 18 degrees. 5 uM 14C-labeled Adenosine and Uridine were added to the tube containing 5 oocytes, and incubated for 1-60 min at different time intervals. Concentration dependent study was carried out at different concentrations of Uridine ranging from 100uM - 4mM. The oocytes were washed 6 times in transport buffer and lysed with 1% sodium dodecyl sulfate and counted in a Liquid scintillation counter.

Confocal images confirmed the ENT1 protein localization to the plasma membrane. The results of the time-dependent study showed that the deletions were able to transport Adenosine and Uridine, while the concentration dependent study showed no major variation in the Km for the substrates, confirming that the extra and intracellular loops in hENT1 are not essential for the transport of neither Adenosine nor Uridine.

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/content/papers/10.5339/qfarf.2011.BMP30
2011-11-20
2020-11-25
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